Polymeric nanocomposites loaded with fluoridated hydroxyapatite Ln3+ (Ln = Eu or Tb)/iron oxide for magnetic targeted cellular imaging

Objective To fabricate polymeric nanocomposites with excellent photoluminescence, magnetic properties, and stability in aqueous solutions, in order to improve specificity and sensitivity of cellular imaging under a magnetic field. Methods Fluoridated Ln3+-doped HAP (Ln3+-HAP) NPs and iron oxides (IOs) can be encapsulated with biocompatible polymers via a modified solvent exaction/evaporation technique to prepare polymeric nanocomposites with fluoridated Ln3+-HAP/iron oxide. The nanocomposites were characterized for surface morphology, fluorescence spectra, magnetic properties and in vitro cytotoxicity. Magnetic targeted cellular imaging of such nanocomposites was also evaluated with confocal laser scanning microscope using A549 cells with or without magnetic field. Results The fabricated nanocomposites showed good stability and excellent luminescent properties, as well as low in vitro cytotoxicity, indicating that the nanocomposites are suitable for biological applications. Nanocomposites under magnetic field achieved much higher cellular uptake via an energy-dependent pathway than those without magnetic field. Conclusion The nanocomposites fabricated in this study will be a promising tool for magnetic targeted cellular imaging with improved specificity and enhanced selection.


Introduction
With the advances in molecular and cellular techniques, research about the diagnosis of disease has been directed toward the underlying molecular and genomic aberrations, rather than toward clinical signs and symptoms alone. Molecular imaging is a developing research discipline aimed at visually characterizing normal and pathologic processes in living organisms at the cellular and molecular levels [1][2][3][4] . Modalities used in molecular imaging includes positron emission tomography, magnetic resonance imaging , single-photon emission computed tomography, optical imaging, and ultrasound 5 . These modalities differ in terms of spatial resolution, temporal resolution, sensitivity in probe detection, depth of signal penetration, availability of biocompatible molecular imaging agents, and cost. Optical imaging, an emerging molecular imaging mode, is based on the detection of photons after their interaction with the tissue. This technique has high sensitivity and spatial resolution, features fast and easy to perform procedures, and requires inexpensive and available optical reporters and dyes for simultaneous imaging of multiple processes 3 . As an optical approach, fluorescence imaging is used for molecular imaging.
As hydroxyapatite (HAP) is the main inorganic component of bones and teeth of humans and animals, it exhibits good biocompatibility in biological applications [6][7][8] . HAP doped with rare earth elements has been used as luminescent materials for biological imaging [8][9][10][11][12][13][14][15][16][17] . Hui et al. 16 used a hydrothermal approach to dope rare earth elements with fluorine ions to fabricate fluoridated HAP nanocrystals, which demonstrated excellent luminescent properties for biological imaging. The quenching of the excited state of rare earth elements is caused by −OH ions in the lattice, which were replaced with F − ions to prepare materials with low vibrational energies for rare earth fluorescent transition 8 . Hui et al. 16 [18][19][20] . Although QDs are suitable for biological imaging, they exhibit toxicity caused by the oxidative degradation of their heavy metal contents, thereby limiting their applications in biological fields [21][22][23] . The toxicity of QDs can only be partly resolved through surface modification or encapsulation in biodegradable NPs [24][25][26][27][28][29] . As an alternative to QDs, fluoridated HAP:Ln 3+ NPs present lower toxicity; these NPs are also inexpensive and feature anti-corrosion properties. Therefore, fluoridated HAP:Ln 3+ NPs can be potentially used for molecular imaging. Targeted molecular imaging of cancer is generally achieved through passive and active targeting [30][31][32][33] . In passive targeting, probes accumulate at tumor sites through an enhanced permeability and retention effect, which can be attributed to two factors [34][35][36] . First, angiogenic tumors produce vascular endothelial growth factor, which hyperpermeabilizes tumorassociated neovasculature and results in the leakage of circulating probes. Second, tumors lack an effective lymphatic drainage system, which causes subsequent probe accumulation 33 . In active targeting, targeting agents, such as ligands, attach on the surface of small particles through various conjugation mechanisms 2,33,37,38 . The targeted probes can recognize and bind to specific ligands unique to cancer cells; subsequently, the probes largely accumulate in cancer cells while minimizing distribution in noncancerous cells adjacent to the targeted tissue. Although active targeting is superior in molecular imaging, it is limited for clinical applications because of patient-to-patient variation in receptor expression levels and non-specific expression of receptors in normal tissues. Unlike active targeting, magnetic targeting utilizes magnetic field to accumulate magnetic probes in the cellular area where the magnet is placed [39][40][41][42][43][44][45][46][47] . Magnetic targeting is a general targeting approach because it is based on physical interactions and not limited by specific receptor expression. Gu et al. 46 fabricated magnetic mesoporous silica nanoparticles (M-MSNs) and found that the internalization of M-MSNs by A549 cancer cells could be accelerated and enhanced under magnetic field; the internalization is mainly through an energydependent pathway, namely, clathrin-induced endocytosis, rather than passive diffusion or magnetic pull-down process.
In this study, a facile method was used to fabricate novel nanocomposites for magnetic targeted cellular imaging of cancer. Fluoridated Ln 3+ -doped HAP (Ln 3+ -HAP) NPs and iron oxides (IOs) can be encapsulated with biocompatible polymers [such as poly(D,L-lactide-co-glycolide), PLGA] via a modified solvent exaction/evaporation technique to prepare polymeric nanocomposites with fluoridated Ln 3+ -HAP/iron oxide. The fabricated composite can be used to improve specificity and sensitivity of cellular imaging through magnetic targeting. In this study, an external magnetic field was used for targeting to allow the nanocomposites to be as close as possible to A549 cancer cells. Most nanocomposites around A549 cells can be internalized by the cells via an energy-dependent pathway. This study utilizes magnetic field to achieve targeted cellular imaging of cancer with fluoridated Ln 3+ -HAP NPs for cancer diagnosis. The scheme of synthesis and applications of fluoridated Ln 3+ -HAP/IOs PLGA nanocomposites is presented in Figure 1.

Preparation of fluoridated Ln 3+ -HAP (Ln = Eu or Tb) NPs
Fluoridated Ln 3+ -HAP NPs were prepared using the protocol described in the literature 8

Preparation of IOs
IOs were fabricated according to the method reported in the literature 48 . Briefly, 0.6275 g of FeSO 4 ·(NH 4 ) 2 SO 4 ·6H 2 O was dissolved in 16 mL of water. The solution was mixed with 0.8 g of NaOH, 8 mL of oleic acid and 8 mL of ethanol under stirring at room temperature. The solution was then added with an aqueous solution of FeSO 4 ·(NH 4 ) 2 SO 4 ·6H 2 O. The mixed reactants were then transferred to a 50 mL sealed autoclave and heated at 180 ℃ for 10 h. The products were collected through centrifugation and washed at least 3 times with cyclohexane and ethanol. Finally, IOs were obtained through drying and stored for further use.

Cell culture
Human cervical HeLa cell line A549 were obtained from the American Type Culture Collection. All cell culture related reagents were purchased from Invitrogen. The cells were grown in DMEM with 10% FBS and 1% penicillin/streptomycin. The fluoridated Ln 3+ -HAP/IOs PLGA nanocomposites containing a 1:1 mass ratio of the fluoridated Eu 3+ -HAP and IOs were used for cell experiments.

Cytotoxicity of nanocomposites
The viability of A549 cells incubated with the nanocomposites was evaluated with MTT assay. Briefly, the cells were seeded in 96well microplates at a density of 5×10 4 cells/mL. After 24 h of cell attachment, the cells were incubated with 10, 20, 40, 80, 150, and 300 µg/mL nanocomposites for 8 and 24 h. The nanocomposites were then removed, and the cells were washed with PBS three times. The wells were washed twice with PBS and added with 10 μL of MTT supplemented with a culture medium. After 4 h of incubation, the culture medium was removed and the precipitate was dissolved in isopropanol. The absorbance of the wells was determined with a microplate reader (VictorШ, Perkin-Elmer) with a wavelength of 570 nm and reference wavelength of 620 nm. Cell viability was calculated with the following equation: where Int s is the absorbance intensity of the cells incubated with the nanocomposites suspension and Int control is the absorbance intensity of the cells incubated with the incubation medium only (positive control).

Magnetic targeted cellular imaging
A549 cells were maintained at 37 ℃ in a culture medium under a humidified condition of 5% CO 2 . On the day prior to treatment, the cells were seeded in a glass bottom dish with a density of 50,000 cells/mL. For magnetic targeted cellular imaging, a cubic permanent magnet (1.3T) was placed under the edge of the dish and the cells were incubated with 250 µg/mL nanocomposites for 2 h at 37 ℃. Control groups were incubated with the nanocomposites at the same concentration for 2 h without the magnetic field. The cells were then washed with PBS, and the nuclei were stained with 4',6-diamidino-2-phenylindole dihydrochloride for 30 min. The stained cells were washed twice with PBS, and cell images were obtained using a confocal laser scanning microscope (CLSM, Zeiss 710 3-channel; Zeiss, Germany) with an excitation wavelength of 405 nm.

Surface morphology
The TEM images of fluoridated Eu 3+ -HAP NPs and IOs are shown in Figure 2A both are well monodispersed. Figure 2C,D represent the XRD patterns of fluoridated Eu 3+ -HAP NPs and IOs, respectively. The XRD image of fluoridated Eu 3+ -HAP NPs shows the absence of impurities in the final products, thereby demonstrating that Eu 3+ has been successfully doped into HAP. The XRD image of IOs displays that the final product is a pure phase of IOs. Figure 3A-C show the TEM images of the fluoridated Eu 3+ -HAP/IOs PLGA nanocomposites with different Eu 3+ -HAP and IOs mass ratios (2:1, 1:1, and 1:2). These nanocomposites are dispersed as individual nanocomposite with a uniform size of about 200 nm in diameter under each ratio. Figure 3D displays the high-resolution TEM image of the nanocomposite at 1:1 mass ratio of fluoridated Eu 3+ -HAP and IOs. Fluoridated Eu 3+ -HAP and IOs can be well encapsulated into PLGA NPs, and the encapsulation does not result in morphological changes. The EDS map in Figure 4 presents the element distribution of the fluoridated Eu 3+ -HAP/IOs PLGA nanocomposites, thereby demonstrating that fluoridated Eu 3+ -HAP and IOs can be well encapsulated into PLGA NPs. The nanocomposites with fluoridated Eu 3+ -HAP and IOs at a mass ratio of 1:1 were used for the following study because of their fluorescence and magnetic property.

Emission spectrum
The emission spectra of the fluoridated Eu 3+ -HAP/IOs PLGA nanocomposites and fluoridated Tb 3+ -HAP/IOs PLGA nanocomposites in water are demonstrated in Figure 5. The emission spectrum of the fluoridated Eu 3+ -HAP/IOs PLGA nanocomposites was determined from 550 to 750 nm under 405 nm excitation (Figure 5A), and the main emission peak of nanocomposites is located at 615 nm. Figure 5B shows the emission spectrum of the fluoridated Tb 3+ -HAP/IOs PLGA nanocomposites under 488 nm exitation, and the main emission peak is located at 548 nm. The spectra of the fluoridated Ln 3+ -HAP/IOs PLGA nanocomposites demonstrate that the strong fluorescence of the nanocomposites remains, despite the encapsulation of fluoridated Ln 3+ -HAP NPs into PLGA NPs. As the nanocomposites are stable with the existence of IOs, they are considered suitable for targeted cellular imaging.

Magnetic property
The magnetic properties of IOs and the fluoridated Eu 3+ -HAP/   Figure 6. Saturation magnetizations are 38.4 and 11.5 emu/g for IOs and the nanocomposites, respectively. The decrease in saturation magnetization for the nanocomposites may be due to the encapsulation of the PLGA matrix. .

In vitro cytotoxicity of nanocomposites
The cytotoxity of the fluoridated Ln 3+ -HAP/IOs PLGA nanocomposites to A549 cells was evaluated using MTT assay to examine their suitability for biological applications. Figure 7 shows that the fluoridated Ln 3+ -HAP/IOs PLGA nanocomposites display low cytotoxicity to A549 cells. The cell viability values are higher than 90% even when the concentration of the nanocomposites reached 300 μg/mL. This finding further confirms the low toxicity of the nanocomposites. Therefore, the fluoridated Ln 3+ -HAP/IOs PLGA nanocomposites could be potentially used for biological applications.

Magnetic targeted cellular imaging
The application of the fluoridated Ln 3+ -HAP/IOs PLGA nanocomposites for magnetic targeted cellular imaging was investigated with CLSM with or without magnetic field. Figure 8 demonstrates the CLSM images of A549 cells incubated with 250 µg/mL fluoridated Eu 3+ -HAP/IOs PLGA nanocomposites at 37 ℃ for 2 h without the magnetic field (Row 1) and under a magnetic field of 1.3T (Row 2). The CLSM images in red channel in Figure 8 show

Conclusion
Fluoridated Ln 3+ -HAP/IOs PLGA nanocomposites were prepared through a modified solvent exaction/evaporation technique. The fabricated nanocomposites show excellent photoluminescence, magnetic properties, and stability in aqueous solutions. Thus, the composites are suitable for targeted cellular imaging. The results of in vitro experiments further confirm that the nanocomposites exhibit low toxicity and can be successfully applied to improve the specificity and sensitivity of cellular imaging under magnetic field. The nanocomposites fabricated in this study will be a promising tool for magnetic targeted cellular imaging with improved specification and enhanced selection.